María López-Pavía

Integrating clinical features and genetic lesions in the risk assessment of patients with chronic myelomonocytic leukemia

Chronic myelomonocytic leukemia (CMML) is a myelodysplastic/myeloproliferative neoplasm with variable clinical course. To predict the clinical outcome, we previously developed a CMML-specific prognostic scoring system (CPSS) based on clinical parameters and cytogenetics. In this work, we tested the hypothesis that accounting for gene mutations would further improve risk stratification of CMML patients. We therefore sequenced 38 genes to explore the role of somatic mutations in disease phenotype and clinical outcome. Overall, 199 of 214 (93%) CMML patients carried at least 1 somatic mutation. Stepwise linear regression models showed that these mutations accounted for 15% to 24% of variability of clinical phenotype. Based on multivariable Cox regression analyses, cytogenetic abnormalities and mutations in RUNX1, NRAS, SETBP1, and ASXL1 were independently associated with overall survival (OS). Using these parameters, we defined a genetic score that identified 4 categories with significantly different OS and cumulative incidence of leukemic evolution. In multivariable analyses, genetic score, red blood cell transfusion dependency, white blood cell count, and marrow blasts retained independent prognostic value. These parameters were included into a clinical/molecular CPSS (CPSS-Mol) model that identified 4 risk groups with markedly different median OS (from >144 to 18 months, hazard ratio [HR] = 2.69) and cumulative incidence of leukemic evolution (from 0% to 48% at 4 years, HR = 3.84) (P < .001). The CPSS-Mol fully retained its ability to risk stratify in an independent validation cohort of 260 CMML patients. In conclusion, integrating conventional parameters and gene mutations significantly improves risk stratification of CMML patients, providing a robust basis for clinical decision-making and a reliable tool for clinical trials.

Rapid Screening of ASXL1, IDH1, IDH2, and c-CBL Mutations in de Novo Acute Myeloid Leukemia by High-Resolution Melting

Recently, many novel molecular abnormalities were found to be distinctly associated with acute myeloid leukemia (AML). However, their clinical relevance and prognostic implications are not well established. We developed a new combination of high-resolution melting assays on a LightCycler 480 and direct sequencing to detect somatic mutations of ASXL1 (exon 12), IDH1 (exon 4), IDH2 (exon 4), and c-CBL (exons 8 and 9) genes to know their incidence and prognostic effect in a cohort of 175 patients with de novo AML: 16 patients (9%) carried ASXL1 mutations, 16 patients had IDH variations (3% with IDH1(R132) and 6% with IDH2(R140)), and none had c-CBL mutations. Patients with ASXL1 mutations did not harbor IDH1, [corrected] or CEBPA mutations, and a combination of ASXL1 and IDH2 mutations was found only in one patient. In addition, we did not find IDH1 and FLT3 or CEBPA mutations concurrently or IDH2 with CEBPA. IDH1 and IDH2 mutations were mutually exclusive. Alternatively, NPM1 mutations were concurrently found with ASXL1, IDH1, or IDH2 with a variable incidence. Mutations were not significantly correlated with any of the clinical and biological features studied. High-resolution melting is a reliable, rapid, and efficient screening technique for mutation detection in AML. The incidence for the studied genes was in the range of those previously reported. We were unable to find an effect on the outcome.

In vitro all-trans retinoic acid sensitivity of acute myeloid leukemia blasts with NUP98/RARG fusion gene

Dear Editor, Retinoic acid receptors (RARs) are nuclear hormone receptors functioning as ligand-activated transcription factors which interact specifically in order to modulate transcription DNA elements. RARs include alpha (RARA), beta (RARB), and gamma (RARG) receptor types. They function as heterodimers with retinoid X receptors (RXRs). Heterodimers formed by RXRA-RARG are necessary for growth arrest and visceral and primitive endodermal differentiation [1, 2]. The RARA is known to be involved in the t(15;17) chromosome translocation uniquely associated with acute promyelocytic leukemia (APL) which generates the RARA/PML fusion [1]. In a few APL cases, RARA is rearranged with partner genes other than PML as a result of variant translocations [3, 4]. In a previous report, we described a novel fusion protein generated by a translocation t(11;12)(p15;q13) that involved the genes NUP98 and RARG in a patient with acute myeloid leukemia (AML) [5]. The involvement of RARG in a chimeric fusion transcript had not been reported previously in human leukemia. Interestingly, the patient blasts displayed morphologic and immunophenotypic features resembling the classical hypergranular subtype of acute promyelocytic leukemia. As reported, the patient achieved complete remission with standard AML chemotherapy followed by autologous peripheral blood transplantation [5]. Two years later, the patient relapsed with the same morphologic, immunophenotypic and molecular features displayed at diagnosis. The patient achieved second complete remission after the combination of all-trans retinoic acid (ATRA) with standard salvage treatment for relapse, but he died due to an infection complication after umbilical cord blood transplantation. Since it has been previously reported that fusion proteins generated with RARB and RARG also confer responsiveness to ATRA in vitro and in vivo [1, 2], we tested the in vitro sensitivity to ATRA of the patient leukemic blasts. Leukemic cells obtained from the bone marrow were resuspended in RPMI1640 medium, supplemented with 15% FCS and antibiotics at a concentration of 10cells/ml. ATRA (Sigma, St Louis,MO, USA) was added at a final concentration of 10 m. The differentiation effect was assessed by analyzing morphological characteristics (nucleus/ cytoplasm ratio, nuclear shape, granulations, and basophilia) on May-Grünwald-Giemsa stained cytospin slides and by multiparametric flow cytometry using the monoclonal antibodies CD10, CD11b, CD13, CD15, CD16, CD33, CD45, CD117, HLADR, and 7-AAD. Serial studies were performed after incubation at 3 and 5 days for all cell cultures. The experiment was performed in triplicate, and the ATRAsensitive NB4 cell line was used as a control. The analysis did not show changes either in morphological features or differentiation patterns measured by immunophenotype, whereas ATRA-treated NB4 cells showed both morphological differentiation and an increase in CD11b expression as expected (Fig. 1). Since ATRAwas given simultaneously with chemotherapy to our patient, we were unable to evaluate the effect of this agent in vivo. The in vitro studies, however, showed that AML with a NUP98/RARG rearrangement is resistant to Jose Cervera and Miguel A. Sanz senior authors contributed equally to this work. E. Such (*) : L. Cordón :A. Sempere : E. Villamón :M. Ibañez : I. Luna : I. Gómez-Seguí :M. López-Pavía :C. Alonso : J. Cervera :M. A. Sanz Hematology Department, Hospital Universitari i Politècnic La Fe, Valencia, Spain e-mail: such_esp@gva.es

WT1 isoform expression pattern in acute myeloid leukemia

WT1 plays a dual role in leukemia development, probably due to an imbalance in the expression of the 4 main WT1 isoforms. We quantify their expression and evaluate them in a series of AML patients. Our data showed a predominant expression of isoform D in AML, although in a lower quantity than in normal CD34+ cells. We found a positive correlation between the total WT1 expression and A, B and C isoforms. The overexpression of WT1 in AML might be due to a relative increase in A, B and C isoforms, together with a relative decrease in isoform D expression.

The Mutational Landscape of Acute Promyelocytic Leukemia Reveals an Interacting Network of Co-Occurrences and Recurrent Mutations.

Preliminary Acute Promyelocytic Leukemia (APL) whole exome sequencing (WES) studies have identified a huge number of somatic mutations affecting more than a hundred different genes mainly in a non-recurrent manner, suggesting that APL is a heterogeneous disease with secondary relevant changes not yet defined. To extend our knowledge of subtle genetic alterations involved in APL that might cooperate with PML/RARA in the leukemogenic process, we performed a comprehensive analysis of somatic mutations in APL combining WES with sequencing of a custom panel of targeted genes by next-generation sequencing. To select a reduced subset of high confidence candidate driver genes, further in silico analysis were carried out. After prioritization and network analysis we found recurrent deleterious mutations in 8 individual genes (STAG2, U2AF1, SMC1A, USP9X, IKZF1, LYN, MYCBP2 and PTPN11) with a strong potential of being involved in APL pathogenesis. Our network analysis of multiple mutations provides a reliable approach to prioritize genes for additional analysis, improving our knowledge of the leukemogenesis interactome. Additionally, we have defined a functional module in the interactome of APL. The hypothesis is that the number, or the specific combinations, of mutations harbored in each patient might not be as important as the disturbance caused in biological key functions, triggered by several not necessarily recurrent mutations.

Screening for IDH mutations in chronic myelomonocytic leukemia

Recent genome-wide sequencing eff orts have led to the identifi cation of a number of new candidate genes involved in the pathogenesis of myeloid neoplasms. Among them, variations in isocitrate dehydrogenase genes ( IDH1 and IDH2 ) have been identifi ed in several hematologic malignancies. IDH1 and IDH2 mutations have been found in 8 – 10% and 15% of patients with acute myeloid leukemia, res pectively, especially in those having normal karyotype (NK-AML). Both gene mutations were recently also reported in myelodysplastic syndromes and chronic myeloproliferative disorders, and were associated with a high rate of leukemic transformation and poor prognosis [1 – 3]. A few studies have been published on somatic mutations of IDH1 and IDH2 in chronic myelomonocytic leukemia (CMML). Th e reported incidence of these mutations in CMML ranged from 0 to 9% [3 – 7], although their prognostic relevance remains uncertain. All reported IDH1 mutations involve the R132 residue, while IDH2 mutations involve either the R140 or the R172 residue. Mutations in IDH decreased affi nity to isocitrate, displaying a neomorphic catalytic activity toward α -ketoglutarate, producing an accumulation of 2-hydroxyglutarate [2,8 – 10]. It is currently believed that these intracellular changes facilitate oncogenic pathways, including activation of hypoxia-inducible factor-1 α (HIF-1 α ). Nevertheless, their relative contribution is still unclear [2].

Prognostic impact of gene mutations in myelodysplastic syndromes with ring sideroblasts

Myelodysplastic syndromes (MDS) are a heterogeneous group of myeloid neoplasms with extremely variable clinical outcome. The latest update of the World Health Organization (WHO) classification (2016) has defined various MDS subtypes on the basis of dysplastic and cytopenic lineages, the prevalence of blasts, the percentage of ring sideroblasts (RS), and the presence of cytogenetic and genetic abnormalities. In this assessment, the category defined by ring sideroblasts, MDS-RS, is subdivided into cases with single-lineage dysplasia (MDS-RSSLD) and cases with multilineage dysplasia (MDS-RSMLD). Moreover, in the presence of cytopenias, dysplasia, and as few as 5% of RS, the MDS-RS category can be established by the identification of a mutation in the SF3B1 gene. Therefore, genetic data like SF3B1 mutation provide diagnostic utility in MDS and probably also relevant prognosis information. In recent years, nextgeneration sequencing (NGS) studies in MDS have found new recurrently mutated genes . Thus, we have utilized these findings for a specific mutational analysis of the MDS-RS subgroup. We studied 122 patients diagnosed of MDS-RS according to the 2016 WHO classification. From them, 80 patients (66%) had been diagnosed by morphology of MDS-RS-SLD and 42 patients (34%) corresponded to MDS-RS-MLD (Supplementary Table 1). The International Prognostic Scoring System (IPSS) was low for 105 patients (86%) and intermediate-1 for 17 patients (14%). According to the Revised IPSS (IPSS-R), 53 patients (43%) were classified as very low risk, 60 patients (50%) as low risk, 8 patients (6%) as intermediate risk, and only 1 patient (1%) as high risk (Supplementary Table 1). The median follow-up was 35 months (95% confidence interval, 6–204 months), and 5 patients (4%) progressed to acute myeloid leukemia (AML, Supplementary Table 2). DNA samples were isolated from bone marrow samples at diagnosis and were obtained with written informed consent in accordance with the Declaration of Helsinki, and the approval of the internal review of Bioethics and Medical Research at the University Hospital La Fe. NGS was based on AmpliSeq chemistry and was performed on an Ion Proton instrument. A custom panel of 39 genes with 659 amplicons was designed and allowed a 98% detection rate for 5% variant frequency at positions with an average sequencing coverage from 1000× to 5000× (Supplementary Methods; Supplementary Table 3). Mutations of CALR exon 9, not included in the original panel, were analyzed by Sanger sequencing. In the statistical analyses P< 0.05 values were considered as statistically significant. With this approach, up to 97% patients (118 out of 122) have been found to have a somatic mutation in at least one gene (Supplementary Tables 4 and 5). As it was expected, the majority of cases carried SF3B1 mutations (106/122, 86.9%), but other mutations were also detected: TET2 (38/122, 31.1%); DNMT3A (21/122, 17.2%); JAK2 (8/122, 6.6%); SRSF2 (6/122, 4.9%); SETBP1 (6/122, 4.9%); EZH2 (5/122, 4.1%); and ZRSR2 (5/122, 4.1%) genes (Fig. 1).

BRAF V600E mutation in adult acute lymphoblastic leukemia

BRAF V600E is a point mutation located in exon 15 of the serine/threonine protein kinase B-Raf ( BRAF ) gene, and was originally discovered to recurrently occur in melanoma [1]. Because of this acquired change, the RAS/RAF/MEK/ ERK/mitogen activated protein kinase (MAPK) pathway is constitutively activated, leading to increased protein kinase activity and abnormal cell proliferation. Th is pathway has been implicated in acute leukemia oncogenesis [2,3], and the BRAF V600E mutation has been reported in association with oncohematological neoplasms [4 – 14]. Virtually all cases of hairy cell leukemia (HCL) show the BRAF V600E mutation [4 – 6]. Albeit at a very low rate, other B-lymphoid neoplasms such as chronic lymphocytic leukemia and multiple myeloma also show this mutation [4,8,9]. By contrast, acute myeloid leukemia seems to lack this alteration [10]. Davidsson et al . [11] found no such mutation in 92 children with acute lymphoblastic leukemia (ALL). However, in another study of 29 children, one patient (3%) with T cell-ALL (T-ALL) carried the BRAF V600E mutation [12]. To our knowledge, two studies have analyzed the presence of the BRAF V600E mutation in adult ALL. Th e study of Lee et al . [13] involved only 12 patients; two of them carried BRAF G468A, but no mutation in BRAF V600E was found. Recently, Ping et al . [14] did not observe mutations in exon 15 of BRAF in 115 adults with ALL using Sanger sequencing. If present, this alteration may have prognostic value, as described for other neoplasms.

Single-Nucleotide Polymorphism Array-Based Karyotyping of Acute Promyelocytic Leukemia

Acute promyelocytic leukemia (APL) is characterized by the t(15;17)(q22;q21), but additional chromosomal abnormalities (ACA) and other rearrangements can contribute in the development of the whole leukemic phenotype. We hypothesized that some ACA not detected by conventional techniques may be informative of the onset of APL. We performed the high-resolution SNP array (SNP-A) 6.0 (Affymetrix) in 48 patients diagnosed with APL on matched diagnosis and remission sample. Forty-six abnormalities were found as an acquired event in 23 patients (48%): 22 duplications, 23 deletions and 1 Copy-Neutral Loss of Heterozygocity (CN-LOH), being a duplication of 8(q24) (23%) and a deletion of 7(q33-qter) (6%) the most frequent copy-number abnormalities (CNA). Four patients (8%) showed CNAs adjacent to the breakpoints of the translocation. We compared our results with other APL series and found that, except for dup(8q24) and del(7q33-qter), ACA were infrequent (≤3%) but most of them recurrent (70%). Interestingly, having CNA or FLT3 mutation were mutually exclusive events. Neither the number of CNA, nor any specific CNA was associated significantly with prognosis. This study has delineated recurrent abnormalities in addition to t(15;17) that may act as secondary events and could explain leukemogenesis in up to 40% of APL cases with no ACA by conventional cytogenetics.

The modular network structure of the mutational landscape of Acute Myeloid Leukemia

Acute myeloid leukemia (AML) is associated with the sequential accumulation of acquired genetic alterations. Although at diagnosis cytogenetic alterations are frequent in AML, roughly 50% of patients present an apparently normal karyotype (NK), leading to a highly heterogeneous prognosis. Due to this significant heterogeneity, it has been suggested that different molecular mechanisms may trigger the disease with diverse prognostic implications. We performed whole-exome sequencing (WES) of tumor-normal matched samples of de novo AML-NK patients lacking mutations in NPM1, CEBPA or FLT3-ITD to identify new gene mutations with potential prognostic and therapeutic relevance to patients with AML. Novel candidate-genes, together with others previously described, were targeted resequenced in an independent cohort of 100 de novo AML patients classified in the cytogenetic intermediate-risk (IR) category. A mean of 4.89 mutations per sample were detected in 73 genes, 35 of which were mutated in more than one patient. After a network enrichment analysis, we defined a single in silico model and established a set of seed-genes that may trigger leukemogenesis in patients with normal karyotype. The high heterogeneity of gene mutations observed in AML patients suggested that a specific alteration could not be as essential as the interaction of deregulated pathways.